The present invention relates to novel chimeric basic fibroblast growth factors and to the enhanced production of such factors (bFGF).
Polypeptide growth factors are hormone-like modulators of cell proliferation and differentiation. Growth factors are responsible for the regulation of a variety of physiological processes, including development, regeneration and wound repair.
In the course of study of these factors, a number have been identified on the basis of the ability of extracts from various tissues, such as brain, pituitary and hypothalamus, to stimulate the mitosis of cultured cells. Numerous shorthand names have been applied to active factors in these extracts, including epidermal growth factor, platelet-derived growth factor, nerve growth factor, hematopoietic growth factor and fibroblast growth factor.
Fibroblast growth factor (FGF) was first described by Gospodarowicz in 1974 (Nature 249: 123-127) as derived from bovine brain or pituitary tissue which was mitogenic for fibroblasts and endothelial cells. It was later noted that the primary mitogen from brain was different from that isolated from pituitary. These two factors were named acidic and basic FGF, respectively, because they had similar if not identical biological activities but differed in their isolectric points. Acidic and basic fibroblast growth factors (recently reviewed by Burgess, W. H., and Maciag, T. Ann. Rev. Biochem. 58: 575-606 (1989)) appear to be normal members of a family of heparin-binding growth factors that influence the general proliferation capacity of a majority of mesoderm- and neuroectoderm-derived cells (Gospodarowicz, D., et al., Nat. Cancer Inst. Mon. 48:109-130 (1978)), including endothelial cells, smooth muscle cells, adrenal cortex cells, prostatic and retina epithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblasts and osteoblasts (Burgess and Maciag, cited above at page 584). Although human melanocytes respond to the mitogenic influences of basic fibroblast growth factor but not acidic FGF most avian and mammalian cell types respond to both polypeptides (ibid.).
In addition to eliciting a mitogenic response that stimulates cell growth, fibroblast growth factors can stimulate a large number of cell types to respond in a non-mitogenic manner. These activities include promotion of cell migration into wound areas (chemotaxis), initiation of new blood vessel formation (angiogenesis), modulation of nerve regeneration (neurotropism), and stimulation or suppression of specific cellular protein expression, extracellular matrix production and cell survival important in the healing process (Burgess and Maciag, cited above, pages 584 to 588).
These properties, together with cell growth promoting action, provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing and in prevention and therapeutic applications for thrombosis, artheriosclerosis, and the like. Thus, fibroblast growth factors have been suggested to promote the healing of tissue subjected to trauma (Davidson, J. M., et al. J. Cell Bio. 100:1219-1227 (1985)), to minimize myocardium damage in heart disease and surgery (U.S. Pat. Nos. 4,296,100 and 4,378,347 to Franco), and to increase neuronal survival and neurite extension (Walicke, P., et al., Proc. Nat. Acad. Sci. USA 83: 3012-3016 (1986)).
Complementary DNA clones encoding human acidic and human and bovine basic fibroblast growth factors have been isolated and sequenced, and the predicted amino acid sequences derived from the complementary DNAs agree with the structures determined by protein sequence analysis (summarized by Burgess and Maciag, cited above, at pages 580-581). The data predict acidic fibroblast growth factor (hereafter referred to as aFGF) to have 155 amino acids (ibid). The gene for basic fibroblast growth factor (hereafter referred to as bFGF) also codes for a 155 residue protein. For both aFGF and bFGF N-terminally truncated forms that exhibit full biologic activity including a 146-amino acid bFGF originally isolated and sequenced (Esch, F., et al, Proc. Nat. Acad. Sci. USA 82 6507-6511 (1985)) and a 131-amino acid form. Analysis of the structures demonstrates a 55% identity between aFGF and bFGF (Burgess and Maciag, cited above at page 581).
Basic fibroblast growth factor may be extracted from mammalian tissue, but this requires several steps even when heparin-linked affinity chromatography is employed (U.S. Pat. Nos. 4,785,079 and 4,902,782 to Gospodarowicz, et al.), and the 146-amino acid species is generally obtained if extraction is done in the absence of protease inhibitors (ibid., column 9, lines 29 to 32). Bovine and human basic fibroblast growth factor cDNA have been expressed in E. coli (Iwane, M., et al., Biochem. Biophys. Res. Commun. 146:470-477 (1987) and Squires, C. H., et al., J. Biol. Chem. 263:16297-16302 (1988)) and S. Cervisiae (Barr, P. J.. et al., J. Biol. Chem. 263: 16471-16478 (1988)). However, reported yields of product are low (see Eur. Pat. Ap. Pub. No. 228,449 to Esch, et al., page 18), and recombinant factors exhibit a marked tendency to undergo thiol-disulfide interchanges promoted by free thiol groups in the protein that result in the formation of disulfide scrambled species (Iwane, cited above).
A number of basic fibroblast growth factor analogues have been suggested. Muteins of bFGF having amino or carboxyl terminal amino acids deleted, amino acids added, cysteine substituted with a neutral amino acid such as serine, or aspartic acid, arginine, glycine, serine, or valine substituted with other acids have been suggested to have enhanced stability (Eur. Pat. Ap. Pub. No. 281,822 to Seno, et al., page 4, lines 1 to 3, and page 6, line 29 to page 7, line 19); the muteins comprise two or three additions, deletions or substitutions, with substitution of serine for cysteine the most preferred substitution (page 7, lines 18 to 23). Arakawa and Fox (Eur Pat. Ap. Pub. No. 320, 148) suggested replacing at least one, and more preferably two, of the cysteines found in natural bFGF with a different amino acid residue to yield a more stable analogue (page 4, lines 44 to 47); serine was illustrated in the Examples (page 13, lines 22 to 23), but alanine, aspartic acid and asparagine were also suggested (page 5, line 26 and page 13, line 25). Similarly, recombinant aFGFs having extraneous bond-forming cysteine replaced with serine, and oxidation-prone cysteine, methionine and tryptophan replaced with alanine, valine, leucine or isoleucine, to yield factors having enhanced or improved biological activity have also been suggested (Eur. Pat. Ap. Pub. No. 319,052 to Thomas Jnr and Linemeyer, page 17, lines 8 to 20).
A bFGF mutein lacking 7 to 46 amino acids from the carboxyl terminus and, optionally, having amino acids replacements was suggested to have improved stability while retaining activity in Eur. Pat. Ap. Pub. No. 326,907 to Seno, et al. (page 2, line 50 to page 3, line 4). Fiddes, et al, (Eur. Pat. Ap. Pub. No. 298723) suggested replacing basic or positively charged residues in the heparin binding domain encompassing residues 128 to 138 with neutral or negatively charged amino acids to produce forms of FGF having reduced heparin binding ability and enhanced potency (page 5, line 45, and page 5, line 54 to page 6, line 16). Bergonzoni, et al., suggested six analogues: 1) M1-bFGF, lacking residues 27 to 32; M2-bFGF, lacking residues 54 to 58; M3-bFGF, lacking residues 70 to 75; M4-bFGF, lacking residues 78 to 83; M5 -bFGF, lacking residues 110 to 120; M5a-bFGF, having the position 128 lysine and the position 129 arginine replaced with glutamine residues; and M6b-bFGF, having the positions 119 and 128 lysines and the positions 118 and 129 arginines replaced by glutamine residues (Eur. Pat. Ap. Pub. No. 363,675, column 6, line 48 to column 7, line 13).
However, new stable and active forms of fibroblast growth factors are increasingly sought to use in the therapies indicated hereinabove.